ES2383574T3 - Polypeptide libraries with a predetermined framework - Google Patents

Abstract

Populations of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence E XXX A XX EI X X LPNLT X XQ X X AFI X KL X DD PSQSSELLSE AKKLNDSQ or AKYAKE XXX A XX EI XX LPNLT X XQ XX AFI X KL X DDPSQSSEL LSEAKKLNDS Q, wherein each X individually corresponds to an amino acid residue which is varied in the population are disclosed. Also populations of polynucleotides, wherein each member encodes a member of a polypeptide population are disclosed. Furthermore, combinations of such polypeptide populations and such polynucleotide populations are disclosed, wherein each member of polypeptide population is physically or spatially associated with the polynucleotide encoding that member via means for genotype-phenotype coupling. Furthermore, methods for selecting a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, isolation of a polynucleotide encoding a desired polypeptide having an affinity for a predetermined target, identifying a desired polypeptide having an affinity for a predetermined target, selecting and identifying a desired polypeptide having affinity for a predetermined target, and production of a desired polypeptide having an affinity for a predetermined target are disclosed.

Description

Polypeptide libraries with a predetermined framework

Field of the Invention

The present invention relates to new populations of polypeptide variants based on a common framework. These populations can among others be used to provide new proteins and binding polypeptides.

Background

Different procedures for the construction of new binding proteins have been described (Nygren PA and Uhlén M (1997) Curr Opin Struct Biol 7: 463-469). One strategy has been to combine library generation and exploration

or selection with respect to desired properties.

Original Affibody® molecules, populations of such molecules and frameworks of such molecules have been described among others in WO 95/19374, the teaching of which is incorporated herein by reference.

In some applications, proteins, polypeptide or Affibody® molecules, populations of such molecules and frameworks with improved properties, such as alkaline stability, low antigenicity, structural stability, susceptibility to chemical synthesis and hydrophilicity are desired.

Alkaline stability

The production of protein pharmaceutical agents and biotechnology reagents requires several stages of purification to enrich with respect to specific product while removing unwanted contaminants. The affinity purification mediated by protein affinity matrices such as monoclonal antibodies and Staphylococcal A protein (SpA) allows efficient purification in one step. However, to make this profitable it is desirable to be able to regenerate the affinity matrices properly. This usually involves a procedure known as site cleaning (CIP), in which agents, often alkaline solutions, are used to elute contaminants.

Alkaline stability with molecular image capture indicators is also required to mark with the most common technetium-99m SPECT nuclide, and to allow some other types of chemical modifications to be made.

Low antigenicity

Protein-based pharmaceutical compounds, such as therapeutic monoclonal antibodies and Affibody® molecules, have the potential to induce unwanted immune responses in humans. The main factors that contribute to immunogenicity are the presence of impurities, protein aggregates, foreign epitopes such as new idiotypes, different Ig allotypes or non-own sequences. In addition, cross-reaction immunoglobulin (Ig) interactions will most likely increase the likelihood of generating a memory immune response mediated by specific T lymphocytes against the protein pharmaceutical product. To minimize the risk of unwanted interaction with the immune system it is desirable to eliminate existing immune epitopes by protein engineering of the pharmaceutical product.

Affibody® molecules are derived from Staphylococcal A protein (SpA), which is a cell wall-associated receptor on the surface of the Gram positive Staphylococcal aureus bacteria. More precisely, SpA is composed of five highly homologous domains that all bind to immunoglobulins of many mammalian species including humans. Each SpA domain interacts with human Ig in two different ways; by direct union with Fcy including IgG1, IgG2 and IgG4 (Langone JJ (1982) Adv Immunol 32: 157-252), or by union with members of the VH3 family (Silverman GJ et al (1992) Int Rev Immunol 9: 57 -78). The common framework of the original Affibody® molecules is identical to the SpA B domain with the exception of the G29A mutation, which was included to increase protein stability and to eliminate a hydroxylamine cleavage site, and the A1V mutation, introduced into a spacer region between domains (Nilsson B et al (1987), Prot Eng 1: 107-113). The amino acid residues in SpA that have been implicated in the interaction with Fcy and VH3 are well known and have been described in the literature (Graille M et al (2000) Proc Natl Acad Sci USA. 97: 5399-5404). A molecular library of different Affibody® molecules was constructed by randomly selecting surface residues on one side of the molecule, including residues known to be involved in the interaction with Fcy, thereby eliminating Fcy affinity.

Structural stability

One of the key factors for the success of protein and peptide pharmaceuticals is protein stability. Proteins that show high structural stability will more functionally withstand chemical modifications and proteolysis during production as well as within the human body. In addition, stability will influence the expiration period of peptide or protein pharmaceutical products as well as the active life of the pharmaceutical or protein product within the human body.

Susceptibility of chemical synthesis

Researchers have traditionally obtained proteins by biological procedures but the chemical synthesis of peptides and small proteins is a potent complementary strategy and is commonly used in structural biology, protein engineering and biomedical research. Chemical protein synthesis offers a fast and efficient way to produce homogeneous proteins without biological contaminants such as DNA impurities and host cell proteins. In addition, flexibility is increased since chemical synthesis allows the incorporation of unnatural amino acids, chemical modifications and introduction of biochemical and biophysical probes. The success of the chemical synthesis of peptides and proteins depends on the amino acid sequence of the molecule in question. Certain amino acid residues show low coupling efficiency, which means that several stages need to be optimized during the synthesis, which is a time-consuming procedure without guaranteed success. In addition, amino acids that are difficult to introduce effectively during chemical synthesis will have a greater negative impact on protein yield the longer the protein sequence.

Increased hydrophilicity

For most applications it is desirable that the peptides and proteins be highly soluble, showing a low tendency to aggregate. Such protein characteristics are especially important when it comes to protein pharmaceuticals. There is a strong positive correlation between protein surface hydrophobicity and low solubility and increased tendency to aggregate.

Description of the invention

It is an object of the present invention to provide a population of polypeptide variants based on new frameworks.

These new frames have several advantages compared to similar known frames, called original frames. The advantages also apply to polypeptides obtained with the use of these new frameworks. These advantages will be discussed in more detail later, but some examples are provided herein. For example, extensive research has been conducted to develop new polypeptide frameworks that show high stability in an alkaline environment. An aspect of alkaline stability is stability against asparagine deamidation. Avoiding this reaction by increased structural rigidity or replacing this residue also provides chemical stability that contributes to obtaining a homogeneous product, for example after production in a fermentation or storage process, in which the deamidation of asparagine contributes greatly to a mixture. heterogeneous that is difficult to separate.

In addition, an improved profile was obtained with respect to low antigenicity (poor binding to IgG) by elimination in the new framework sequence of the remaining affinity with respect to immunoglobulins, mainly mediated by VH3.

In addition, the new frames have been genetically engineered so that they show high structural stability with respect to an easily folded alpha helical structure, high melting temperature and suppression of sites known to be directed to proteolysis.

The original Affibody® molecule frameworks contain several amino acids that have been shown to reduce the speed and success of chemical synthesis. In order to have an efficient Affibody® protein production process, that is to say high yield and high production, numerous amino acids were exchanged for residues with properties more compatible with chemical synthesis.

To improve hydrophilicity and solubility, hydrophobic moieties on the surface of the Affibody® molecule framework have been replaced by more hydrophilic amino acids.

Another object of the present invention is to provide a population of polynucleotides.

Another object of the present invention is to provide a combination of a population of polypeptides and a population of polynucleotides.

A further object of the present invention is to provide a method for selecting a desired polypeptide that has an affinity for a predetermined target of a population of polypeptides.

Another object is to provide a method for isolating a polynucleotide encoding a desired polypeptide that has an affinity for a predetermined target.

Another object is to provide a method for identifying a desired polypeptide that has an affinity for a predetermined target.

A further object is to provide a method for selecting and identifying a desired polypeptide that has an affinity for a predetermined target.

A related object is to provide a process for producing a desired polypeptide that has an affinity for a predetermined target.

The populations and methods according to the invention allow the provision (including production and evaluation) of agents with an affinity for a predetermined target, through the provision of a polypeptide characterized by specific binding with the predetermined target.

It is also possible to provide polypeptides that bind to a predetermined target that shows little or no nonspecific binding.

It is also possible to provide polypeptides that bind to a predetermined target that can be easily used as a moiety in a fusion polypeptide.

In addition, it is possible to provide polypeptides that bind to a predetermined target that solves one or more of the known problems experienced with existing antibody reagents.

In addition, it is possible to provide polypeptides that bind to a predetermined target that are susceptible to use in therapeutic and / or diagnostic applications.

It is also possible to provide polypeptides that bind to a predetermined target that are readily prepared by chemical peptide synthesis.

In addition, the invention allows the identification of polypeptides that bind to a predetermined target that shows improved stability against known agents that bind to the same target.

It is also possible to provide polypeptides that bind to a predetermined target that show low antigenicity when used in vivo in a mammal and / or that show improved biodistribution after administration to a mammal.

These and other objects are fulfilled by the different aspects of the invention as claimed in the appended claims.

In a first aspect the invention provides a population of polypeptide variants based on a common framework, each polypeptide in the population comprising the framework amino acid sequence EXXXAXXEIX XLPNLTXXQXXAFIXKLXDDPSQSSELLSE AKKLNDSQ, (SEQ ID NO: 1).

or in some cases more preferably

In the previous sequences each X individually corresponds to an amino acid residue that is varied in the population.

The population consists of a large number of variants of such polypeptide molecules. In this context a large number means a population comprising at least 1 x 104 unique polypeptide molecules, at least 1 x 106, at least 1 x 108, at least 1 x 1010, at least 1 x 1012 or at least 1 x 1014 molecules bound polypeptides.

35 However, it is necessary to use a group that is large enough to provide the desired population size. The "population" described herein can also be indicated as "library."

It has been previously indicated that each X corresponds individually to an amino acid residue that varies. This means that each X can be an amino acid residue independent of the identity of any other residue indicated as X in the sequence. In the framework amino acid sequence the different varied X amino acids can be selected from the 20 naturally occurring amino acid residues such that any of these 20 naturally occurring amino acid residues can be present at the corresponding X position in any given variant. The selection of amino acid residues in each position is more or less random. It is also possible to limit the group from which the different varied amino acid residues are selected to 19, 18, 17, 16 or less of the 20 naturally occurring amino acid residues. Variability in different positions can be adjusted individually, between one, 45 which means without random selection, up to 20 amino acids. Random introduction of a smaller subset of amino acids can be obtained by careful selection of introduced deoxyribonucleotide bases, for example, T (A / C) C codons can be introduced to obtain a random introduction of serine or tyrosine at a given position in the polypeptide chain. Similarly, codons (T / C / A / G) CC can be introduced to obtain a random introduction of phenylalanine, leucine, alanine and valine in one position

given in the polypeptide chain. The person skilled in the art is aware of many alternatives of combinations of deoxyribonucleotide bases that can be used to obtain different combinations of amino acids at a given position in the polypeptide chain. The set of amino acids that may appear at a given position in the polypeptide chain can also be determined by the introduction of trinucleotides during oligonucleotide synthesis, instead of one deoxyribonucleotide base each time.

Polypeptides comprising the amino acid sequences of the framework provided above are new Affibody® molecules. As such, they are derived from Staphylococcal Protein A (SpA). In this context "derivative" does not mean that the polypeptides themselves in any way necessarily originate directly from SpA. Instead it means that the framework has a sequential and structural similarity with a SpA domain, in which amino acids are conserved in the hydrophobic core of the three helical beam protein.

Different modifications of, and / or additions to, the polypeptides that constitute the population according to the invention can be made to adapt the polypeptides to the specific intended use, without departing from the scope of the present invention. Such modifications and additions are described in more detail below, and may comprise additional amino acids comprised in the same polypeptide chain, or markers and / or therapeutic agents that are chemically conjugated or otherwise bound to the polypeptides that constitute the population. In some embodiments, additional amino acid residues at the C-terminus may be preferred. These additional amino acid residues may play a role in the binding of the polypeptide, but other purposes, for example related to one or more of the production, purification, stabilization, coupling or detection of the polypeptide, may serve equally well. Such additional amino acid residues may comprise one or more amino acid residues added for chemical coupling purposes. An example of this in the addition of a cysteine residue at the first or last position in the polypeptide chain, that is at the N or C terminal end. A cysteine residue can also be introduced to be used for chemical coupling by replacing other amino acids on the surface of the protein domain, preferably in a part of the surface that is not involved in target binding. Such additional amino acid residues may also comprise a "tag" for purification or detection of the polypeptide, such as a hexahistidyl (His6) tag, or a "myc" tag or a "FLAG" tag for interaction with specific tag antibodies. The person skilled in the art is aware of other alternatives.

The "additional amino acid residues" discussed above may also constitute one or more polypeptide domains with any desired function, such as another binding function, an enzymatic function, a metal ion chelating function or a fluorescent function or mixtures thereof.

In a second aspect, the invention provides a population of polynucleotides. Each polynucleotide in this population encodes a member of a polypeptide population described above.

In a third aspect the invention provides a combination of a population of polypeptides according to the invention and a population of polynucleotides according to the invention in which each member of the polypeptide population is physically or spatially associated with the polynucleotide encoding that member by means for genotype-phenotype coupling. This physical or spatial association will be more or less strict, depending on the system used.

The means for genotype-phenotype coupling can comprise a phage display system. Phage display systems are well known to those skilled in the art and are described, for example, in Smith GP (1985) Science 228: 1315-1317 and Barbas CF et al (1991) Proc Natl Acad Sci USA 88: 7978 -7982.

In addition, the means for genotype-phenotype coupling can comprise a cell surface presentation system. The cell surface presentation system may comprise prokaryotic cells, such as Gram + cells or eukaryotic cells, such as yeast cells. The cell surface presentation systems are well known to those skilled in the art. Prokaryotic systems are described, for example, in Francisco JA et al (1993) Proc Natl Acad Sci U S A 90: 10444-10448 and Lee SY et al (2003) Trends Biotechnol 21: 45

52. The eukaryotic systems are described, for example, in Boder ET et al (1997) Nat Biotechnol 15: 553-557 and Gai SA et al (2007) Curr Opin Struct Biol 17: 467-473.

In addition, the means for genotype-phenotype coupling can comprise a presentation system without cells. The cellless presentation system may comprise a ribosome presentation system, an in vitro compartmentalization presentation system, a cis presentation system or a microbead presentation system. Ribosome presentation systems are well known to those skilled in the art and are described, for example, in Mattheakis LC et al (1994) Proc Natl Acad Sci USA 91: 9022-9026 and Zahnd C et al (2007) Nat Methods 4: 269-279. In vitro compartmentalization systems are well known to those skilled in the art and are described, for example, in Sepp A et al (2002) FEBS Lett 532: 455-458. Cis presentation systems are well known to those skilled in the art and are described, for example, in Odegrip R et al (2004) Proc Natl Acad Sci U S 101: 2806-2810. Microbead presentation systems are well known to those skilled in the art and are described, for example, in Nord O et al (2003) J Biotechnol 106: 1-13.

In addition, the means for genotype-phenotype coupling can comprise a system without presentation such as the fragment-protein complementation assay (PCA). PCA systems are well known to those skilled in the art and are described, for example, in Koch H et al (2006) J Mol Biol 357: 427-441.

In a fourth aspect the invention provides a method for selecting a desired polypeptide having an affinity for a predetermined target of a population of polypeptides, comprising the steps of:

(to)

 provide a population of polypeptides as described above;

(b)

 bringing the population of polypeptides into contact with the predetermined target under conditions that allow specific interaction between the target and at least one desired polypeptide having an affinity for the target; Y

(C)

 selecting, based on said specific interaction, the at least one desired polypeptide from the remaining population of polypeptides.

This procedure is subsequently referred to as the selection procedure according to the invention.

Step (a) may comprise the preparatory steps of providing a population of nucleotides and expressing said population of polynucleotides to produce said population of polypeptides. The means for producing a population of polypeptides varies depending on the presentation system used and examples of such media can be found in the genotype-phenotype references above. Each member of said population of polypeptides used in the screening method according to the invention can be physically associated with the polynucleotide encoding that member by means of genotype-phenotype coupling. The means for genotype-phenotype coupling can be one of those discussed above.

Step (b) comprises the steps of bringing the population of polypeptides into contact with the predetermined target under conditions that allow specific interaction between the target and at least one desired polypeptide having an affinity for the target. The series of applicable conditions is determined by the robustness of the target, the robustness of the presentation system and by the desired properties of the interaction with the target. For example, a specific procedure may be desired to separate the interaction such as acidification at a predetermined pH. The person skilled in the art knows what experiments are required to determine the appropriate conditions.

Step (c) comprises the selection of at least one polypeptide. The means for selecting the desired polypeptide from the remaining population, based on the specific interaction between the predetermined target and at least one desired polypeptide having affinity for the target varies depending on the presentation system used and can be found in the genotype-phenotype references above. . For example, in vitro presentation selection systems are cell-free, unlike systems such as phage display and protein fragmentation compartmentalization assay.

In a fifth aspect the invention provides a method for isolating a polynucleotide encoding a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

-

selecting said desired polypeptide and the polynucleotide encoding it from a population of polypeptides using the selection method according to the invention based on the use of said frameworks and

-

isolate the separated polynucleotide in this way that encodes the desired polypeptide.

This procedure is subsequently referred to as the isolation procedure according to the invention.

The separation of the polynucleotide from the polypeptide can be performed differently depending on the presentation system used for selection. For example, in cellless presentation systems such as cis presentation and ribosome presentation, the polynucleotide or the corresponding mRNA are recovered through effective elution of the polypeptide using means described in the genotype-phenotype references above.

The polynucleotide isolation can be performed by different procedures depending on the presentation system used for the selection. In most of the selection systems commonly described, for example the protein fragment complementation assay, the polynucleotide can be isolated directly by specific PCR amplification using appropriate oligonucleotides. Exceptionally, as in the presentation of ribosomes, the polynucleotide can be isolated from the corresponding mRNA using reverse transcription. The various means for polynucleotide isolation can be found in the genotype-phenotype references above.

In a sixth aspect the invention provides a method for identifying a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

-

isolating a polynucleotide encoding said desired polypeptide using the isolation procedure according to the invention; Y

-

sequencing the polynucleotide to establish by deduction the amino acid sequence of said desired polypeptide.

Sequencing of the polynucleotide can be carried out in accordance with conventional procedures well known to those skilled in the art.

In a seventh aspect the invention provides a method for selecting and identifying a desired polypeptide having an affinity for a predetermined target of a population of polypeptides, comprising the steps of:

(to)

 synthesize each member of the polypeptide population in a separate vehicle or bead;

(b)

 select or enrich the vehicles or beads based on the interaction of the polypeptide with the predetermined target; Y

(C)

 Identify the polypeptide by protein characterization methodology. In step (c), it is for example possible to use mass spectrometric analysis.

This procedure is subsequently referred to as the selection and identification procedure according to the invention.

In an eighth aspect the invention provides a process for producing a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

-

selecting and identifying a desired polypeptide using the selection procedure according to the invention or the selection and identification procedure according to the invention using said frameworks, and

-

producing said desired polypeptide.

This procedure is subsequently called the production process according to the invention.

In the production process according to the invention the production can be carried out using recombinant expression of a polynucleotide encoding the desired polypeptide. Production can also be carried out using chemical synthesis of the desired de novo polypeptide.

In a ninth aspect the invention provides a process for the production of a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

(a1) isolating a polynucleotide encoding said desired polypeptide using the isolation procedure according to the invention: or (a2) backtracking an identified polypeptide using the selection and identification procedure according to the invention; Y

(b) expressing the isolated polynucleotide in this way to produce said desired polypeptide,

in which stage (b) is performed after stage (a1) or stage (a2).

The expression of the polynucleotide can be carried out in any suitable expression host known to the person skilled in the art such as but not limited to bacterial cells, yeast cells, insect cells or mammalian cells.

Expressions such as "binding affinity for a predetermined target", "binding with a predetermined target" and the like refer to a property of a polypeptide that can be measured directly through the determination of affinity constants, ie the amount of polypeptide that associates and dissociates at a given antigen concentration. Different procedures can be used to characterize molecular interaction, such as, but not limited to, competition analysis, equilibrium analysis and microcalorimetric analysis and real-time interaction analysis based on surface plasmon resonance interaction (for example using a Biacore® instrument ). These procedures are well known to those skilled in the art and are described, for example, in Neri D et al (1996) Tibtech 14: 465-470 and Jansson M et al (1997) J Biol Chem 272: 8189-8197.

The inventors of the present invention have discovered that a polypeptide that binds to a predetermined target, obtained by any of the aforementioned methods can show one or more surprising advantages, compared to known polypeptides that bind to the same target, while they retain the ability of previously known polypeptides to bind to the target. Non-limiting examples of such advantages are the following:

 A polypeptide, which binds to a predetermined target and is obtained by any of the procedures

mentioned above, it comprises fewer amino acid residues that could cause problems,

such as low yield and success rate, in chemical synthesis of the polypeptide sequence, such as

asparagine, arginine, aspartic acid and methionine. A polypeptide, which binds to a predetermined target and is obtained by any of the procedures

previously mentioned, it comprises less amino acid residues that confer hydrophobicity of

surface. This implies fewer problems with low solubility and aggregation. Without wishing to be linked to the

In theory, it is currently believed that the most hydrophilic characteristics act to shift the biodistribution of the polypeptide after administration to a host, from a hepatobiliary route (excretion through the liver) to a more desired renal route (excretion through the kidneys). A polypeptide, which binds to a predetermined target and is obtained by any of the above-mentioned procedures, comprises fewer amino acid residues that are associated with polypeptide stability problems, such as methionine, asparagine and the asparagine-proline dipeptide. Methionine is susceptible to oxidation, asparagine is susceptible to deamidation and the asparagine-proline bond is susceptible to acidic cleavage, and thus contribute to the non-homogeneity of the final product. A polypeptide, which binds to a predetermined target and is obtained by any of the aforementioned methods, lacks amino acid residues that, in a similar sequence context, have been found to increase interaction with immunoglobulins that contain a variable chain domain. heavy of VH3 (Silverman GJ (1992) mentioned above). Without wishing to be bound by theory, it is currently believed that the replacement of such amino acid residues in a polypeptide, which bind to a predetermined target and are obtained by any of the methods mentioned above, reduces the antigenicity of the polypeptide after administration thereof. to a guest

Among the advantages of the framework of the invention, alkaline stability, low antigenicity, structural stability, improved properties for chemical synthesis and / or improved hydrophilicity are among the most important.

The polypeptides, which bind to a predetermined target and are obtained by any of the aforementioned procedures, can be used as detection reagents, capture reagents, separation reagents, diagnostic agents for in vivo or in vitro diagnosis, as therapeutic agents by themselves or as means to direct other therapeutic and / or diagnostic agents to the predetermined target. Procedures that use the in vitro polypeptides according to the invention can be performed in different forms, such as microtiter plates, protein matrices, biosensor surfaces, tissue sections and so on.

The modifications discussed above for the polypeptides that constitute the population according to the invention are also applicable to the polypeptides obtained by any of the aforementioned procedures.

Polypeptides according to the invention can be produced by any known means, including chemical synthesis or expression in different prokaryotic or eukaryotic hosts, including transgenic plants and animals.

The invention will now be illustrated in detail through the description of experiments performed accordingly. The following examples should not be construed as limiting. In the examples, reference is made to the attached figures. The invention is limited exclusively as defined by the appended claims.

Brief description of the figures

Figure 1 shows results obtained from DC measurements, using a Jasco J spectropolarimeter

810. The variable temperature measurement of His6-ZTNF-a: 3230 was performed at 0.5 mg / ml in PBS buffer. The absorbance was measured at 221 nm at 20 to 80 ° C, with a temperature slope of 5 ° C / min. A cell with an optimal path length of 1 mm was used. The melting temperature (Tm) of His6-ZTNF-a: 3230 was determined from the measurement of the variable temperature. Figure 2 shows an overview of the selection described in Example 4. The selection was made on two different tracks, one with a high target concentration (track 1) and one with a low target concentration (track 2). Target concentrations are provided for each track and cycle as well as the number of washes (in parentheses). Figure 3 is an overlay representation of two DC spectra before (full line) and after (dashed line) of heating His6-Z04674 at 96 ° C. Figure 4 shows the result of a Biacore analysis of polypeptide variants; Sensograms obtained after sequential injection of His6-Z04777 (full rectangles), His6-Z04687 (full triangles), His6-Z04665 (open triangles), His6-Z04674 (black line), His6-Z04781 (gray line) and execution buffer (open rectangles) on immobilized Dynazyme. The response (in UR) was plotted against the time (s). Figure 5 shows elution profiles of analytical HPLC for polypeptides with the sequence maESEKYAKEMRNAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK (SEQ ID NO: 10) in the synthesis step of amino acid residues 18 to 58. A) Synthesis performed on polystyrene resin in positions 22-23, 41-42, 45-46 and 53-54. B) Synthesis of conventional peptide in polystyrene resin without pseudoprolines. Figure 6 shows elution profiles of analytical HPLC for polypeptides with the sequence maESEKYAKEMRNAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK (SEQ ID NO: 10) at the stage of synthesis of amino acid residues 1 to 58 (A) and 10 to 58 (B). A) Synthesis performed on polystyrene resin using pseudoprolines in positions 22-23, 41-42, 45-46 and 53-54. B) Synthesis of conventional peptide in polystyrene resin without pseudoprolines. Figure 7 shows the elution profiles of analytical HPLC for polypeptides with the sequence A) AEAKYAKEMW IAWEEIRNLP NLNGWQMTAF IAKLLDDPSQ SSELLSEAKK LNDSQAPKC (SEQ ID NO: 11) (according to the invention) and B) AENKFNKEMW IAWEEIRNQQQQKLQQQKLKLQLQKKDLQLQQKKDKPDLLQKDWPKLWAQLQQKKDWLKQLQKWPKLWKIQLQLWKQKPKDWLQQWPKLQKKDWPKLQLQLWKQKPKLWKWPKDWLQQKKDWLQLKKDWPKLWKQKKDWLWKDWLWKDWLWAQLQKWLWKPKLWKWPKDWLWKWKWKDWLWAWKWAWKWAWKWG No.: 12) (for comparison).

Examples

Example 1 - Construction of a combinatorial polypeptide library

5 A combinatorial polypeptide library was constructed essentially as described in Grönwall C et al (2007) J Biotechnol 128: 162-183, by PCR amplification of a 123 nucleotide template oligonucleotide with certain degenerate codons (5'-GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC-3 ') (SEQ ID NO: 38) encodes helices 1 and 2 of protein Z derived from protein A of

10 Staphylococcus aureus (Nilsson et al (1987), mentioned above), with the point mutations N3A, F5Y, N6A, N23T and S33K. PCR amplification was performed using primers AFFI-1364 and AFFI-1365 with an Xho I site and a Sac I site, respectively, underlined in Table 1.

The resulting gene fragment encoding the library was restricted with Xho I and Sac I. Subsequently, the gene fragment encoding the library was ligated into a phagemid vector restricted by Xho I and Sac I adopted to

15 phage display called pAY2016, essentially based on the phaffid vector pAffi1 (Grönwall C et al (2007), mentioned above), in phase with amino acid residues 41-58 of the Z protein, which encode helix 3 with point mutations A42S, N43E, A46S and A54S. Helix 3 was constructed by hybridizing the two complementary oligonucleotides AFFI-1333 and AFFI-1334 (Table 1).

The resulting library vector was introduced by electroporation into Escherichia coli strain RR1iM15 (Ruther U 20 (1982) Nucl Acids Res 10: 5765-5772), producing a 2.4 x 1010 member library.

Preparation of phage repertoires from the library was performed using conventional procedures involving the auxiliary phage M13K07 (New England Biolabs, Beverly, MA, United States), routinely producing phage titres of approximately 1011 cfu per ml of culture.

Table 1. List of oligonucleotides

Name

Sequence 5’-3 ’

AFFI-1333

AGCTCTGAATTACTGAGCGAAGCTAAAAAGCTAAATGATAGC CAGGCGCCGAAAGTAGACTAC (SEQ ID NO: 34)

AFFI-1334

GTAGTCTACTTTCGGCGCCTGGCTATCATTTAGCTTTTTAGCTTCGCTCAGTAATTCAGAGCT (SEQ ID NO: 35)

AFFI-1364

AAATAAATCTCGAGGTAGATGCCAAATACGCCAAAG (SEQ ID NO: 36)

AFFI-1365

TAAATAATGAGCTCTGGCTTGGGTCATC (SEQ ID NO: 37)

Example 2 - Selection of phage display and characterization of human HER2 binding polypeptide variants

Summary

Biotinylated HER2 protein is used as a target in phage display selections using the library

30 constructed in Example 1. Selections are made using a variety of conditions to maximize the probability of obtaining molecules that have a high affinity for HER2. After elution of the selected phages, the corresponding expressed proteins are tested for affinity for HER2 in an ELISA preparation. Positive clones are identified and sequenced, and the predicted amino acid sequences of the corresponding polypeptides and their HER2 binding motifs are deduced, resulting in a large number of

35 sequences of HER2 binding molecules.

Biotinylation of HER2

Lyophilized human HER2 protein (R&D Systems, No. 1129-ER) is dissolved in PBS (2.68 mM KCI, 1.47 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4, pH 7.4) at a final concentration of 10 mg / ml Sulfo-NHS-LC-Biotin EZ-link (Pierce, No. 21335) is dissolved in water at a final concentration of 1 mg / ml and a molar excess of 5 and

40 30 times at 500 µg of HER2 in a total volume of 0.5 ml. The mixtures are incubated at room temperature (RT) for 30 minutes. Unbound biotin is removed by dialysis against PBS using a dialysis cassette (Slide-A-Lyser, 10 kDa; Pierce).

Phage Display Selection

In total, five cycles of selection are carried out, using increasingly stringent conditions, such as decreasing HER2 concentration and increasing wash numbers. Three initial cycles are carried out, mainly with the intention of establishing an appropriate selection protocol. Selection is then carried out for two more cycles using the combinations of selection buffer, target concentration and solid support listed in Table 2.

Table 2: Selection conditions for HER2 selection

Sample Name

Selection Buffer Supplement Target Concentration (nM) Streptavidin in pearls (! G)

Cycle 4

TO Jelly twenty 100

B

Jelly 10 100

C

BSA 5 100

D

BSA 2.5 100

Cycle 5

TO Jelly 10 fifty

B

Jelly 5 fifty

C

BSA one fifty

D

BSA 0.5 fifty

All tubes and beads (Streptavidin M-280 Dynabeads®, No. 112.06; Dynal) used in the procedure of

10 selection are preblocked in TPBSB (5%) (Tween 20 0.05%, bovine serum albumin (BSA), sodium azide 0.02% in PBS) or gelatin (0.5%) for at least 30 minutes to TA.

The selection solutions (1 ml) contained biotinylated human HER2, phages, sodium azide (0.02%), Tween 20 (0.05%) and BSA (3%) or gelatin (0.1%) according to the Table 2, and are prepared in PBS. Phages are incubated with biotinylated human HER2 target at 4 ° C for three days for Cycle 4 and for one day for Cycle 5, followed by 15 1 hour incubation with stirring at RT. Selection samples are transferred to streptavidin beads blocked for 15 minutes under stirring at RT. The beads are washed 10 times with 1 ml of selection buffer (i.e. TPBSB (3%) (0.05% Tween 20, 3% bovine serum albumin (BSA), 0.02% sodium azide in PBS) or GT 0.1 (0.1% gelatin, 0.1% Tween 20 and 0.02% sodium azide in PBS)), followed by 10 washes with PBS in which the penultimate wash is performed for 5 minutes. Phages are eluted with 1000 µl of 50 mM glycine-HCl, pH 2.2, 20 for 10 minutes at RT, followed by immediate neutralization with 900 µl of PBS supplemented with 100 µl of 1 M Tris-HCl, pH 8.0, or eluted with 1000 µl of trypsin (2 mg / ml) for 30 minutes at RT followed by the addition of 1000 µl of aprotinin (0.4 mg / ml). Eluted phages (3/4 of the volume) are used to infect 50 ml of E. coli RR1iM15 cells in the logarithmic phase (Rüther, 1982, mentioned above) after each selection cycle. After 30 minutes of incubation with gentle agitation and 30 minutes with vigorous agitation at 37 ° C, the cells

25 are centrifuged and the sediment is dissolved in a smaller volume and spread on TYE plates (15 g / l agar, 10 g / l tryptone water (Merck), 5 g / l yeast extract, 3 g / l NaCl supplemented with 2% glucose and 100 µg / ml ampicillin) and finally incubated overnight at 37 ° C.

Preparation of phage repertoire

Plate cells are resuspended in TSB medium (tryptic soybean broth 30 g / l) and the concentration

Cellular is determined by measuring the optical density at 600 nm assuming that OD600 = 1 corresponds to 5 x 108 cells / ml. The cells are inoculated (excess of approximately 100 times of cells compared to eluted phages) in 100 ml of TSB + YE medium supplemented with 2% glucose and 100 µg / ml ampicillin and grown at 37 ° C at approximately OD 600 = 0, 5-0.7. Then, 10 ml is transferred to a new flask and infected by 10-fold molar excess of M13K07 auxiliary phage (New England Biolabs, No. NO315S) and incubated for 30 minutes

35 with low agitation. The cells are sedimented at 2000 g for 10 minutes and resuspended in 100 ml of TSB + YE medium supplemented with isopropyl 1-D-1-thiogalactopyranoside (IPTG) 100 µM, kanamycin 50 µg / ml and ampicillin 100 µg / ml and allowed to grow overnight at 100 rpm and 25 ° C. A part of the resuspended cells is stored at -80 ° C as a glycerol pool.

The overnight culture is centrifuged at 2500 g for 10 minutes and the phages in the supernatant precipitate.

40 adding 1/4 of the volume of precipitation buffer (20% PEG / 2.5M NaCl) and incubate on ice for 1 hour. The precipitated phages are sedimented by centrifugation at 10,000 g at 4 ° C for 30 minutes, resuspended in

20 ml of PBS and then the precipitation procedure is repeated. The phages are finally resuspended in 1 ml of PBS and filtered through a 0.45 µm filter.

Selection, washing and elution solutions are evaluated after each selection cycle. The phage solutions are diluted in sterile water in a microtiter plate and 100 µl of RR1iM15 E. coli cells are added in the logarithmic phase to each phage dilution. After 20 minutes of incubation at RT, 5 µl of each titration is transferred to a TYE plate and incubated overnight at 37 ° C. The resulting colonies are counted and titres (cfu / ml) are calculated.

HER2 binding ELISA analysis

The clones of the final selection cycles are expressed and screened for binding activity with HER2 using an ELISA preparation as described in Example 4 below (but using HER2 as the target protein), or as described below. Randomly selected colonies are expressed in 96-well deep plates by inoculating each colony in 1 ml of TSB + YE medium supplemented with 100 µg / ml ampicillin and 1 mM IPTG and allowed to grow for 18-24 hours at 37 ° C. After incubation, repeated plates are made by transferring a small fraction of each culture to 96-well plates with 15% glycerol for storage at -20 ° C.

The remaining cells are pelleted by centrifugation at 3000 g for 10 minutes, resuspended in 400 µl of 0.05% PBS-T (PBS supplemented with 0.05% Tween 20) and frozen at -80 ° C. The frozen samples are thawed in a water bath and the cells are sedimented at 3700 g for at least 20 minutes. Supernatants containing expressed molecules are collected and used in ELISA.

Medium area microtiter plates (Costar, No. 3690) are coated overnight at 4 ° C with 50 µl of HSA at a concentration of 6 µg / ml in ELISA coating buffer (Sigma, No. 3041). The wells are blocked with 100 µl of blocking buffer (2% skimmed milk powder in PBS) for 2 hours at RT. After removing the blocking buffer, 50 µl of the prepared proteins are added to the wells and the plates are incubated for 1.5 hours at RT. The supernatants are discarded and biotinylated HER2 is added at a concentration of 0.5-10 µg / ml in 0.05 PBS-T to the wells and incubated for 1.5 hours. The bound complexes are detected with horseradish peroxidase, conjugated streptavidin (HRP, Dako, No. P0397) diluted 1: 5000 in PBS-T 0.05, and incubated for 1 hour at RT. 50 µl of ImmunoPure® TMB substrate (Pierce, No. 34021) is added to the wells and plates are treated according to the manufacturer's recommendations. The absorbance of the wells is read at 450 nm in a Tecan Ultra 384 ELISA reader (Tecan) and evaluated using Magellan v software. 5.0 (Tecan). Prior to the addition of each new reagent, four washes are performed with PBS-T 0.05.

Based on the results of this experiment, clones are selected for sequencing as described below.

Sequencing of positive ELISA clones

PCR fragments of selected colonies are amplified using appropriate oligonucleotides. The sequencing of amplified fragments is performed using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's recommendations and the appropriate biotinylated oligonucleotide. Sequencing reactions are purified by binding with paramagnetic beads coated with Dynabeads® REGEN ™ streptavidin using a Magnatrix 8000 instrument (Magnetic Biosolutions) and finally analyzed in ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems).

Plasmid subcloning pAY1448

DNA that encodes specific HER2 molecules and selected in the expression vector pAY1448 is subcloned to create His6-labeled monomeric molecules expressed as MGSSHHHHHHLQ- [Z #####] - VD (His6-Z #####), in which Z ##### indicates an identified member of the starting population of variant molecules. Plasmids containing inserts are purified from 2 ml overnight culture of E. coli RR1iM15 cells in TSB supplemented with 100 µg / ml ampicillin using Qiagen Mini Kit (Qiagen) according to the manufacturer's recommendations.

DNA from the selected molecules in the expression vector pAY1448 is subcloned by cloning of Accl-Notl PCR adhesive ends using the appropriate pairs of PCR primers.

The expression vector pAY1448 is digested in two stages at 37 ° C for 4 hours using Accl and Notl in buffer NEB4 and NEB3 (New England Biolabs), respectively, and dephosphorylated with calf intestinal alkaline phosphatase (CIAP; Fermentas) for 1 hour at 37 ° C. The cleaved plasmid and fragments are purified by QIAquick PCR purification kit (Qiagen) according to the manufacturer's recommendations.

The PCR products are hybridized and ligated into dephosphorylated pAY1448 and digested with AccI-NotI for 1 hour at RT using T4 DNA ligase (5 units / µl; Fermentas). Aliquots of the ligaments in E. coli BL21 cells (DE3) are introduced by electroporation. The cells are plated on tryptose blood agar (TBAB) based plates supplemented with 50 µg / ml kanamycin and incubated overnight at 37 ° C. Positive clones are first checked for inserts with PCR scanning and then analyzed for correct sequences as described above.

Expression and purification of His6-labeled polypeptides

Selected molecules are expressed, all subcloned into pAY1448 as described above, in E. coli BL21 (DE3) as fusions with a terminal His6 N marker and purified by IMAC. A colony of each molecule is used to inoculate 5 ml of TSB medium supplemented with 50 µg / ml kanamycin. The cultures are allowed to grow overnight at 37 ° C. The next day, 50 µl of each culture are inoculated separately to 100 ml of TSB + YE medium supplemented with 50 µg / ml kanamycin in a 1 liter flask. The cultures are allowed to grow at 100 rpm at 37 ° C to an OD600 of 0.7-1, after which IPTG is added to a final concentration of 0.5 mM and the cells are incubated at RT overnight at 100 rpm . The cultures are collected by centrifugation at 8000 g for 5 minutes and the sediments are stored in a freezer until protein preparation.

His6-labeled proteins are purified by IMAC under denatured conditions using 1.5 ml Ni-NTA Superflow columns (Qiagen). The buffer is exchanged to PBS using PD-10 columns (GE Healthcare).

Protein concentration is determined using A280 and the BCA Protein Assay Reagent Kit (Pierce) as recommended by the manufacturer. Protein purity is analyzed by SDS-PAGE stained with Coomassie Blue

R.

Biosensor analysis of the affinity of molecules selected by human HER2

A biosensor analysis is performed on a Biacore2000 instrument (GE Healthcare) with human HER2 immobilized by amine coupling in the carboxylated dextran layer on the surface of a CM-5 microplate (for research use; GE Healthcare) according to the manufacturer recommendations Surface 1 on the microplate is activated and deactivated and is used as a reference cell during injections. The selected molecules, expressed and purified as described above, are diluted in HBS-EP (GE Healthcare) to 25 nM and injected at a constant flow rate of 25 µl / min for 10 minutes, followed by dissociation in HBS-EP for 30 minutes The surfaces are regenerated with two injections of 25 mM HCl.

Example 3 - Cloning, production and evaluation of melting temperature and in vitro antigenicity of original shell variants and of the invention

Summary

The present example describes the cloning, production and evaluation of original frame variants and of the invention. It is believed that introduced shell mutations improve various properties of polypeptide molecules, such as antigenicity, hydrophilicity and alkaline and structural stability. Therefore, different molecules were evaluated with respect to melting temperature and in vitro antigenicity and the results showed that the molecules of the invention had increased melting temperatures and presented lower in vitro antigenicities (lower binding with IgG) compared to the original molecules.

Polypeptide cloning

For the original constructs, DNA sequences encoding specific molecules for Tumor Necrosis Factor alpha (TNF-a), HER2, insulin, Taq polymerase and Platelet-derived Growth Factor Receptor beta (PDGF-R1) were amplified (Table 3). ) by PCR in two individual reactions each (PCR1 and PCR2). Primer pairs AFFI-267 / AFFI-1014 and AFFI-1015 / AF-FI-270 (Table 4) were applied, which encoded parts of the AccI restriction site at the 5 ′ ends in PCR1 and PCR2, respectively. To prepare the plasmid molds, the bacteria that harbored the plasmid DNA were grown overnight in TSB medium, supplemented with 50 µg / ml kanamycin. Cells were pelleted by centrifugation and plasmids were prepared using Spin QIAprep Miniprep Kit (Qiagen).

For constructions of the invention, PCR amplification (PCR1 and PCR2) of nucleotide sequences encoding modified molecules of the invention was performed using partially overlapping oligonucleotides as templates (AFFI-1320-AFFI-1323, AFFI-1326 and AFFI-1327) or using vector with original construction and oligonucleotides containing relevant mutations (AFFI-69, AFFI-70, AFFI-1151 and AFFI-1152) (Table 4). Primer pairs AFFI-1328 / AFFI-1331 and AFFI-1329 / AFFI-1330, which encode parts of the Accl restriction site at the 5 ′ ends, were included in the PCR reactions.

The PCR reactions were amplified using Pfu Turbo DNA polymerase (Stratagene, No. 600854-52) according to a conventional PCR protocol and the PCR products were analyzed by 1% agarose gel electrophoresis.

Z variants binding to PDGF-R1 were generated using oligonucleotides with various codons and a PCR-based mutagenesis technique. The PCR fragments obtained were ligated into an expression vector cleaved using In-Fusion technology (Clontech, No. 639607).

To generate DNA fragments containing upstream and downstream AccI adhesive ends for all constructs, direct and reverse nucleotide chains were separated from the PCR1 and PCR2 products using magnetic streptavidin beads. After 30 minutes of incubation at RT in continuous rotation, the beads were washed with wash buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2, 10 mM DTT) and heated at 95 ° C for 5 minutes. The supernatant containing the non-biotinylated fragments was collected and heated at 95 ° C, followed by stepwise cooling at 25 ° C for 30 minutes to hybridize the DNA strands.

The DNA fragments encoding binding molecules were subsequently ligated to TA, for 2 hours or overnight, in an expression vector treated with CIP (calf intestinal alkaline phosphatase) and purified, previously digested with restriction enzyme AccI. The constructions achieved were MGSSHHHHHHLQ [Z #####] - VD, MGSSLQ- [Z #####] - VDC (for ZPDGF-R1: 2465) or M- [Z #####] - C ( for ZPDGF-R1: 3358).

The ligaments were transformed into electrocompetent TOP10 E. coli cells and plated as described in Example 2. Bacterial colonies that harbored newly constructed plasmids were screened by PCR and the inserted DNA sequences were verified as described in Example 2

Verified plasmids were prepared as described above.

Polypeptide expression

Cultures of E. coli BL21 (DE3) transformed with relevant plasmids were inoculated in 800 ml of TSB-YE medium supplemented with 50 µg / ml kanamycin and 0.3 ml / l antifoam agent (Breox FMT 30) and allowed to grow at 37 ° C to an OD600 of about 2. Protein expression was then induced by adding 1M IPTG to a final concentration of 0.5 mM. Cultures were performed using the Greta multifermenting system (Belach). Cultures were collected 5 hours after induction by centrifugation at 15900 x g for 20 minutes. The supernatants were discarded and the cell pellets were collected and stored at -20 ° C. The level of protein expression was determined using SDS-PAGE and ocular inspection of the stained gels.

Purification of expressed polypeptides

His6-tagged proteins were purified as follows: Settled bacterial cells were suspended that contained His6-labeled polypeptides soluble in His GraviTrap binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole and 40 U / ml Benzonase®) and broke by ultrasonication. After clarification, the supernatants were loaded onto His GraviTrap (GE Healthcare) columns previously equilibrated with His GraviTrap binding buffer. After washing the columns with 10 column volumes (VC) of His GraviTrap wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole), the polypeptides were eluted with 3 VC of His GraviTrap elution buffer ( 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole).

The proteins without His6 label were purified as follows: Sedimented bacterial cells that harbored ZPDGF-R1: 2465-Cys or ZPDGF-R1: 3358-Cys were suspended in 20 mM Tris-HCl, pH 7.1. To break the cells and release the intracellular content, the cell suspensions were heated at 85 ° C for 3 minutes. The lysates were clarified by centrifugation followed by filtration and loaded into 25 ml of Q Sepharose FF (GE Healthcare) packed in a XK26 column (GEHealthcare), previously equilibrated with 20 mM Tris-HCl, pH 7.1. After washing the column with 5 VC of 20 mM Tris-HCl, pH 7.1, the bound proteins were eluted with a linear gradient of 0-0.5 M NaCl in 20 mM Tris-HCl, pH 7.1, during 10 VC The flow rate was 5 ml / min and the 280 nm signal was monitored. Fractions containing ZPDGF-R1: 2465-Cys or ZPDGF-R1: 3358-Cys were identified by SDS-PAGE analysis. The relevant fractions were pooled and the pH adjusted to 8.0 by the addition of 1M Tris-HCl, pH 8.0, to a final concentration of 50 mM. C-terminal cysteine in the constructs was reduced by adding DTT at 20 mM, followed by incubation at 40 ° C for 3 minutes. After reduction, acetonitrile (ACN) was added to a final concentration of 5% and ZPDGF-R1: 2465-Cys or ZPDGF-R1: 3358-Cys was loaded into 1 ml of Resource 15 RPC (GE Healthcare) columns, previously equilibrated with RPC A buffer (0.1% TFA, 5% ACN, 95% water). After column washing with 10 VC of RPC A buffer, bound proteins were eluted with a linear gradient of RPC B Buffer 0-40% (0.1% TFA, 80% ACN, 20% water). The flow rate was 1 ml / min and the 280 nm signal was monitored. Fractions containing ZPDGF-R1: 2465-Cys or pure ZPDGF-R1: 3358-Cys were identified by SDS-PAGE analysis and pooled.

To allow lyophilization of the proteins, the buffer was exchanged to 10 mM ammonium hydrogen carbonate buffer, pH 8.0 or 10 mM ammonium acetate buffer, pH 6.0, using disposable PD-10 desalination columns (GE Healthcare). The lyophilization buffer was selected with respect to the isoelectric point of relevant proteins. Finally, His6-ZTNF-a: 185, His6-ZHER2: 342, His6-ZInsulin: 810, His6-binding polypeptides

ZTaq: 1154, ZPDGF-R1: 2465-Cys, His6-ZHER2: 2628, His6-ZTaq: 3229, His6-ZTNF-a: 3230, His6-ZInsulin: 3232 and ZPDGF-R1: 3358-Cys were lyophilized using an instrument Christ Alfa 2-4 LSC and stored at 4 ° C until use (Table 5). Free C-terminal cysteine was blocked using N-ethylmaleimide (NEM) according to the manufacturer's recommendations (Pierce).

Analysis of purified polypeptides

The concentration of polypeptide solutions was determined by measuring the absorbance at 280 nm using a NanoDrop® ND-1000 Spectrophotometer. Proteins were further analyzed with SDS-PAGE and LC-MS.

For SDS-PAGE analysis, approximately 10 µg of polypeptide was mixed with LDS and DTT Sample Buffer (45 mM final concentration), incubated at 70 ° C for 15 minutes and loaded into 4-12% Bis-Tris Gels NuPAGE®. The gels were processed with MES SDS Execution Buffer in a Novex Mini Cell using the SeeBlue® Plus2 intended standard as molecular weight marker and PageBlue ™ Protein Staining Solution for staining.

To verify the identity of the polypeptides, LC / MS analyzes were analyzed using an Agilent 1100 LC / MSD system, equipped with API-ESI and a simple quadruple mass analyzer. After exchange of buffers, the protein samples were diluted in lyophilization buffer to a final concentration of 0.5 mg / ml and 10 µl were loaded on a Narrow-Bore Zorbax 300SB-C8 column (2.1 x 150 mm, 3.5 µm) at a flow rate of 0.5 ml / min. The proteins were eluted using a linear gradient of 10-70% solution B for 30 minutes at 0.5 ml / min. The separation was performed at 30 ° C. The ionic signal and absorbance at 280 and 220 nm were monitored. The molecular weights of the purified proteins were determined by ionic signal analysis.

Determination of melting temperature (Tm)

Lyophilized polypeptides were dissolved in PBS at a final concentration of approximately 0.5 mg / ml and stored on ice. DC analysis was performed on a Jasco J-81 0 spectropolarimeter in a cell with an optical path length of 1 mm. In variable temperature measurements, the observance was measured at 221 nm at 20 to 80 ° C, with a temperature slope of 5 ° C / min. Melting temperatures (Tm) were calculated for the polypeptides tested by determining the midpoint of the transition in the representation of DC versus temperature.

The modified polypeptide molecules according to the invention had increasing melting temperatures compared to the original molecules (Table 6). In Figure 1, the fusion curve obtained for His6-ZTNF-a: 3230 (TNF-a specific polypeptide of the invention) is shown.

In vitro antigenicity ELISA (serum IgG binding analysis)

The general conditions for the ELISA were as follows: ELISA assays were performed in 96-well plates of medium area. The volumes used were 50 µl per well for all incubations except for the block in which 100 µl were used. Coating was performed overnight at 4 ° C in coating buffer (15 mM Na2CO3 and 35 mM NaHCO3) and all other incubations were performed at room temperature. Dilution of primate serum and detection antibodies in PBS + 0.5% casein was performed. All washes were performed using an automatic ELISA Scan Washer 300, in which each well was washed four times with 175 µl of wash buffer (PBS-T; Tween 20 0.05% in 1 x PBS) per wash.

ELISA plate wells were coated with 2 µg / ml of His6-ZTNF-a binding polypeptides: 185, His6

ZHER2: 342, His6-ZInsulin: 810, His6-ZTaq: 1154, ZPDGF-R1: 2465-Cys, His6-ZHER2: 2628, His6-ZTaq: 3229, His6-ZTNF-a: 3230, His6-ZInsulin: 3232 and ZPDGF-R1: 3358-Cys-NEM. ZHER2: 342 was used as a standard. After coating, the wells were washed twice with tap water and blocked with PBS + 0.5% casein. The plate was emptied and a series of dilutions 2 times of a group of primate serum of mono cinomolgus (Macaca fascicularis; obtained from the Swedish Institute for the Control of Infectious Diseases) was added to the wells. The series of assessments began with a 1/100 dilution and ended at 1/102400. Dilution was performed directly in the 96-well plate. After incubating for 1 hour with the primate serum group, the plate was washed and a goat anti-human HRP-Ig antibody was added in 1/5000 dilution for detection. After 50 minutes of incubation with the detection antibody, the plate was washed and the substrate was added. Equal volumes of the two components were mixed in the ImmunoPure® TMB kit, and 50 µl was added per well. Subsequently, the plate was incubated in the dark for 12 minutes and the reaction was stopped by adding 50 µl of stop solution (2M H2SO4). Absorbance at 450 nm was recorded using an ELISA reader. As a negative control, PBS + 0.5% casein was used instead of the primate serum group.

To evaluate the results and obtain a VAT value that represents the level of primate Ig molecules that bind to the polypeptide, the GraphPad Prism 5 program was used. Sample values were added, with the background OD values subtracted, a a mold based on a non-linear regression formula XY (sigmoid dose response). A dilution value for OD 0.3 was obtained from the formula and the VAT values were calculated by setting the conventional dilution value at 100 and relating all samples to 100. A value below 100 indicates a reduced capacity of polypeptide tested to bind with immunoglobulins compared to the ZHER2: 342 molecule used as a positive control.

The molecules of the invention showed less potential to bind to immunoglobulins compared to original molecules (Table 7). The results are shown as in vitro antigenicity (VAT) values, and a reduced in vitro antigenicity (IgG binding) is read as a reduction in the VAT value.

Table 3. List of binding polypeptide sequences

Name

Amino acid sequence

ZTNF-a: 185

VDNKFNKELGWAIGEIGTLPNLNHQQFRAFILSLWDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 5)

ZHER2: 342

VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 6)

ZInsulin: 810

VDNKFNKEKYMAYGEIRLLPNLNHQQVMAFIDSLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 7)

ZTaq: 1154

VDNKFNKEKGEAWEIFRLPNLNGRQVKAFIASLYDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 8)

ZPDGF-R1: 2465

VDNKFNKELIEAAAEIDALPNLNRRQWNAFIKSLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 9)

Table 4. List of oligonucleotides

Name

Sequence 5’-3 ’

AFFI-069

GTGAGCGGATAACAATTCCCCTC (SEQ ID NO: 13)

AFFI-070

CAGCAAAAAACCCCTCAAGACCC (SEQ ID NO: 14)

AFFI-115

CAGCAAAAAACCCCTCAAGACCC (SEQ ID NO: 15)

AFFI-267

AGATAACAAATTCAACAAAG (SEQ ID NO: 16)

AFFI-270

CTACTTTCGGCGCCTGAGCATCATTTAG (SEQ ID NO: 17)

AFFI-1014

ACTTTCGGCGCCTGAGCATCATTTAG (SEQ ID NO: 18)

AFFI-1015

ATAACAAATTCAACAAAGAA (SEQ ID NO: 19)

AFFI-1043

ACTTTCGGCGCCTGAGAATCATTTAGCTTTTTA (SEQ ID NO: 20)

AFFI-1044

CTACTTTCGGCGCCTGAGAATCATTTAGCTTTTTA (SEQ ID NO: 21)

AFFI-1143

AGATGCCAAATACGCCAAAGAAATGCGAA (SEQ ID NO: 22)

AFFI-1144

ATGCCAAATACGCCAAAGAAATGCGAA (SEQ ID NO: 23)

AFFI-1151

CCCAAGCCAAAGCTCTGAATTGCTATCAGAAGCTAAAAAGC (SEQ ID NO: 24)

AFFI-1152

GCTTTTTAGCTTCTGATAGCAATTCAGAGCTTTGGCTTGGG (SEQ ID NO: 25)

AFFI-1320

AGATGCCAAATACGCCAAAGAAAAGGGGGAGGCGGTGGTT GAGATCTTTAGGTTACCTAACTTAACCGGGAGGCAAGTGAA GGCCTTCATCGCGAAATTATA (SEQ ID NO: 26)

AFFI-1323

CTACTTTCGGCGCCTGGCTATCATTTAGCTTTTTAGCTTCGCTCAGTAATTCAGAGCTC TGGCTTGGGTCATCCCATAATTTAAGGATGAAGGCCCGAAATT (SEQ ID NO: 27)

AFFI-1326

AGATGCCAAATACGCCAAAGAAAAGTATATGGCGTATGGTGAGATCCGGTTGTTACCT AACTTAACCCATCAGCAAGTTATG GCCTTCATCGATAAATTAGT (SEQ ID NO: 28)

AFFI-1327

CTACTTTCGGCGCCTGGCTATCATTTAGCTTTTTAGCTTCGCTCAGTAATTCAGAGCTC TGGCTTGGGTCATCCACTAATTT ATCGATGAAGGCCATAACTT (SEQ ID NO: 29)

(continuation)

Name

Sequence 5’-3 ’

AFFI-1328

AGATGCCAAATACGCCAAAG (SEQ ID NO: 30)

AFFI-1329

ATGCCAAATACGCCAAAGAA (SEQ ID NO: 31)

AFFI-1330

CTACTTTCGGCGCCTGGCTATCATTTAG (SEQ ID NO: 32)

AFFI-1331

ACTTTCGGCGCCTGGCTATCATTTAG (SEQ ID NO: 33)

Table 5. List of polypeptides tested

Diana

Designation Variant

TNF-alpha

His6 -ZTNF-a: 185 Original

TNF-alpha

His6 -ZTNF-a: 3230 Invention

HER2

ZHER2: 342 Original

HER2

His6 -ZHER2: 342 Original

HER2

His6 -ZHER2: 2628 Invention

Insulin

His6 -ZInsulin: 810 Original

Insulin

His6 -ZInsulin: 3232 Invention

Taq polymerase

His6 -ZTaq: 1154 Original

Taq polymerase

His6 -ZTaq: 3229 Invention

PDGF-R1

ZPDGF -R1: 2465-Cys Original

PDGF-R1

ZPDGF -R1: 3558-Cys Invention

Table 6. Determined Tm values of the polypeptides tested

Diana

Designation Variant Tm (ºC)

TNF-alpha

His6 -ZTNF-a: 185 Original 53

TNF-alpha

His6 -ZTNF-a: 3230 Invention 60

HER2

His6 -ZHER2: 342 Original 63

HER2

His6 -ZHER2: 2628 Invention 69

Insulin

His6 -ZInsulin: 810 Original 42

Insulin

His6 -ZInsulin: 3232 Invention 48

Taq polymerase

His6 -ZTaq: 1154 Original 46

Taq polymerase

His6 -ZTaq: 3229 Invention fifty

PDGF-R1

ZPDGF -R1: 2465-Cys-NEM Original 42

PDGF-R1

ZPDGF -R1: 3558-Cys-NEM Invention 42

Table 7. VAT values of the polypeptides tested

Diana

Designation Variant VAT value

TNF-alpha

His6 -ZTNF-a: 185 Original 38

TNF-alpha

His6 -ZTNF-a: 3230 Invention twenty-one

HER2

His6 -ZHER2: 342 Original 99

HER2

His6 -ZHER2: 2628 Invention 14

Insulin

His6 -ZInsulin: 810 Original 43

Insulin

His6 -ZInsulin: 3232 Invention 16

Taq polymerase

His6 -ZTaq: 1154 Original 26

Taq polymerase

His6 -ZTaq: 3229 Invention 18

PDGF-R1

ZPDGF -R1: 2465-Cys-NEM Original 35

PDGF-R1

ZPDGF -R1: 3558-Cys-NEM Invention 3

Example 4 - Selection and characterization of phage display of Dynazyme binding polypeptide variants

5 Dynazyme Biotinylation

The DNA polymerase of the Dynazyme II target protein (Dynazyme) of the species Thermus brockianus (Finnzymes, No. F-501 L) was biotinylated using a 10 x molar excess of Sulfo-NHS-LC-biotin No-weight ™ (Pierce, No. 21327) in accordance with the manufacturer's protocol. The buffer was changed by dialysis using Slide-a-lyzer dialysis cassette (Pierce, 10K, 0.5-3 ml) to PBS before biotinylation and to TKMT (10 mM Tris-HCl, 50 mM KCI, 1.5 mM MgCl2 , Triton X

10 100 0.1%, pH 8.8) after biotinylation to remove unbound biotin.

Phage Display Selection

Selection was made against biotinylated Dynazyme using the population of polypeptides of the invention (Example 1). Two approaches were used for selection; one with a high target concentration (lane 1) and one with a low target concentration (lane 2). Four selection cycles were performed. New repertoires of

15 phages between each cycle. For overview and selection details, see Figure 2.

The phage library repertoire was precipitated with PEG / NaCl twice as described in Example 2 and dissolved in TKMT supplemented with 0.1% gelatin (TKMTg). Phages were pre-incubated with streptavidin coated beads (SA beads, Dynabeads® M-280) for 30 minutes at RT. The beads used in the selection procedure and all the tubes were preblocked in TKMTg.

20 The target concentrations and the number of washes in each cycle are presented in Figure 2. The buffer used for selection and washes was TKMTg. The preselected phages were incubated with the biotinylated target for 17 hours at 4 ° C followed by 3 hours at RT during the first cycle and 3 hours or 1 hour, respectively, during the following cycles. Subsequently, phage particles were transferred to preblocked SA beads, 5 mg (first cycle track 1), 3.5 mg (first cycle track 2) or 0.5 mg (all other cycles) and incubated for 10 minutes with

25 agitation Next, the beads were washed and the phage particles eluted with a low pH buffer as described in Example 2. The eluted phages were used to infect E. coli RR1iM15 cells in the logarithmic phase after each selection cycle. After 20 minutes of incubation at 37 ° C, the cells were collected by centrifugation. The sediment was dissolved in a small volume of TBS-YE, spread on TYE plates and incubated overnight at 37 ° C. Phage repertoires were prepared between each cycle

30 essentially as described in Example 2. Phage particle titres and yields were calculated after each selection cycle. The yield of phage particles (phage particles outside / phage particles inside) increased for each cycle (except the second), indicating an enrichment of the target binding clones.

ELISA analysis of Dynazyme binding polypeptides

35 Clones obtained after the last selection cycle were randomly selected and used for periplasmic protein expression in a 96-well plate format as described in Example 2. Supernatants containing soluble polypeptide variants fused with ABD were tested for binding with target in an ELISA as follows. Potential binding polypeptides were expressed as AQLE- [Z #####] - VDYV- [ABD] -SQKA (ABD = the GA3 albumin binding domain of Streptococcus sp. G148, Kraulis et al (1996) FEBS Lett 378 (2): 190-194), in which Z ##### indicates individual variants of the polypeptide population of the invention.

Medium area microtiter wells (Costar, No. 3690) were coated with 50 µl of Dynazyme 2-3 µl / ml in ELISA coating buffer. The wells were blocked with 100 µl of TKMT supplemented with 0.5% casein (Sigma) (TKMT-casein) for 1 hour at RT. After removal of the blocking solution, 50 µl of supernatants were added to the wells and the plates were incubated for 1.5 hours at RT. Captured polypeptide variants were detected by adding a primary antibody and then a secondary one. The primary antibody, an affinity purified polyclonal rabbit Ig against Z variants, was diluted 1: 5000 in TKMT-casein and incubated for 1.5 hours at RT. The secondary antibody, a goat a-rabbit-HRP Ig (DakoCytomation, No. P0448), was diluted 1: 5000 in TKMT-casein and incubated for 1 hour at RT. The plates were washed four times with TKMT before incubation with the antibodies and the developing solution.

Plates were developed as described in Example 2 and read at 450 nm on an ELISA spectrophotometer. All plates were prepared with relevant negative and positive controls as well as with a blank in which TKMT was used instead of periplasmic supernatant. In total, 1080 randomly selected clones were screened in ELISA for their binding with Dynazyme. Positive clones and some clones with low absorbance values were selected for sequencing.

ELISA positive polypeptide sequencing

Individual clones were subjected to sequencing according to Example 2. Eleven unique binding polypeptides found positive in ELISA scanning were found. Some of the clones appeared in several copies. In addition, several clone sequences with lower ELISA values were identified.

Subcloning of polypeptides in plasmid pA Y1448

Fifteen unique polypeptides were subcloned as monomers in the expression vector pAY1448 to provide a His6 N terminal marker (as described in Example 2) using plasmids prepared as templates for PCR of adhesive ends. Subcloning was performed as described in Example 3.

Polypeptide Purification

The following text describes the purification of fifteen monomeric His6-labeled polypeptides, namely His6-Z04665, His6-Z04672, His6-Z04674, His6-Z04678, His6-Z04687, His6-Z04767, His6-Z04770, His6-Z04775, His6-Z04777 , His6-Z04777, His6-Z04778, His6-Z04779, His6-Z04780, His6-Z04781 and His6-Z04899. Sedimented bacterial cells that harbored His6-labeled molecules soluble in His GraviTrap ™ binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole and 25 U / ml Benzonase®) were suspended and broken by ultrasonication. The sonicated cell suspensions were heated using hot water (95 ° C) until the temperature of the suspensions stabilized at approximately 90 ° C for 5 minutes. After clarification by centrifugation, the supernatants were loaded onto His GraviTrap (GE Healthcare) columns previously equilibrated with His GraviTrap binding buffer. After washing the columns with 5 VC of His GraviTrap binding buffer and 5 VC of His GraviTrap wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole), the polypeptides were eluted with 3 VC of buffer. His GraviTrap elution (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole).

The polypeptide variants were further purified by reverse phase chromatography (RPC). Acetonitrile (ACN) was added to a final concentration of 2% in the eluted fractions of His GraviTrap. The samples were loaded on a 3 ml RESOURCE ™ RPC column (GE Healthcare), previously equilibrated with RPC A buffer (0.1% trifluoroacetic acid (TFA), 2% ACN, 98% water). After washing the column with 5 VC of RPC A buffer, the bound protein was eluted with a linear gradient of 20 VC of RPC B buffer 0-50% (0.1% TFA, 80% ACN, 20% water) . The flow rate was 5 ml / min and the 280 nm signal was monitored. Fractions containing pure polypeptides were identified by SDS-PAGE analysis and pooled.

The buffer of the purified polypeptides was replaced with 50 mM Tris-HCl, pH 8.8, by size exclusion chromatography on disposable PD-10 Desalination Columns (GE Healthcare).

Twelve of the fifteen polypeptide variants were successfully purified: His6-Z04665, His6-Z04672, His6-Z04674, His6-Z04687, His6-Z04770, His6-Z04775, His6-Z04776, His6-Z04777, His6-Z04778 Z04780, His6-Z04781 and His6-Z04899.

Analysis of purified polypeptides

The concentration of polypeptide solutions was determined by measuring the absorbance at 280 nM using a NanoDrop 'ND-1000 Spectrophotometer. Proteins were further analyzed with SDS-PAGE and LC-MS.

For analysis by SDS-PAGE, polypeptide solution was mixed with LDS Sample Buffer (Invitrogen) and incubated at 40 ° C for 10 minutes. 10 µg of each polypeptide variant was loaded into 4-12% Bis-Tris NuPAGE® Gels (Invitrogen). The gels were processed with MES SDS Execution Buffer (Invitrogen) in an XCell SureLock ™ Mini Cell (Invitrogen) using Novex® Clear Pre-Stained Protein Pattern (Invitrogen) as molecular weight marker and PhastGelTM Blue R Protein Staining Solution (GE Healthcare ) for staining.

5 To verify the identity of the polypeptide variants, LC / MS analyzes were performed using an Agilent 1100 LC / MSD system, equipped with API-ESI and single quad mass analyzer. Protein samples were diluted in 50 mM Tris-HCl, pH 8.8, at a final concentration of 0.5 mg / ml and 10 µl were loaded on a Zorbax 300SB-C18 column (4.6 x 150 mm, 3 , 5 µm) (Agilent) at a flow rate of 1 ml / min. Solution A contained 0.1% TFA in water and solution B contained 0.1% TFA in ACN. Proteins were eluted using a linear gradient of 22 minutes of

10 solution B from 15% to 65% at 1 ml / min. The separation was performed at 30 ° C. The ionic signal and absorbance at 280 and 220 nm were monitored. The molecular weights of the purified proteins were verified by ionic signal analysis.

The purity of the polypeptide variants was determined to be greater than 95% according to the SDS-PAGE and CL / MS analyzes.

15 Determination of melting temperature (Tm)

Purified polypeptide variants were diluted in 50 mM Tris-HCl, pH 8.8, to a final concentration of 0.5 mg / ml. Circular dichroism (DC) analysis was performed on a Jasco J-81 0 spectropolarimeter in a cell with an optical path length of 1 mm. In the variable temperature measurements, the absorbance was measured at 221 nm from 20 ° C to 90 ° C, with a temperature slope of 5 ° C / min. The fusion temperatures of polypeptide (Tm) are

20 calculated by determining the midpoint of the transition in the representation of DC versus temperature. For results, see Table 8.

Table 8. Tm of Dynazyme binding polypeptide variants

Designation

Tm (ºC)

His6-Z04665

38

His6-Z04672

33

His6-Z04674

35

His6-Z04687

60

His6-Z04770

44

His6-Z04775

Four. Five

His6-Z04776

55

His6-Z04777

58

His6-Z04778

43

His6-Z04780

65

His6-Z04781

66

His6-Z04899

39

Heat stability analysis

The ability to fold back into the original helical alpha structure after being subjected to heat was a required property of the polypeptide variants described above. To investigate the structural reversibility, two DC spectra were obtained per sample at 20 ° C. Between the two measurements, the samples were heated to 96 ° C. The samples were kept at 96 ° C for 2 minutes, and then cooled to 20 ° C. Similar DC spectra before and after heating would show that a sample was structurally

30 reversible. Three of twelve polypeptide variants analyzed were negatively affected by heat treatment, while it was shown that nine polypeptide variants recovered their helical alpha structure completely. A typical overlap of two DC spectra is shown before and after heating in Figure 3.

Biacore union analysis

The interactions between monomeric Z variants labeled with 12 His6 selected according to the invention and Dynazyme were analyzed in the Biacore instrument (GE Healthcare). The target protein was immobilized in a flow cell in the carboxylated dextran layer of a CM5 microplate surface (GE Healthcare). Immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using acetate pH 5.5. A flow cell surface was activated on the microplate and deactivated for use as a target during analyte injections. The analytes, that is to say polypeptide variants diluted in HBS-EP (GE Healthcare) run buffer at a concentration of 10 µM, were injected in random order in duplicates at a flow rate of 10 µl / min for 5 minutes. After 10 minutes of dissociation, the surfaces were regenerated with an injection of 0.05% SDS. The results were analyzed in BiaEvaluation software (GE Healthcare). The white surface curves were subtracted from the ligand surface curves. The analysis showed an interaction for 5 of the polypeptide variants with the immobilized Dynazyme, as outlined in Figure 4.

Example 5 - Comparative study of chemical synthesis of a polypeptide of a population according to the invention

Summary

In the experiments that comprise this example, solid phase peptide synthesis (SPPS) of polypeptides from populations according to the invention is described, and compared with the synthesis of a polypeptide based on the original framework. Mutations introduced in four positions, ie [N23T], [A42S], [A46S] and [A54S], allowed to use an alternative synthesis strategy with pseudoproline precursors with the simplified abbreviation Fmoc-Xxx-Yyy-OH. Using pseudoprolines in three or four of the positions described above, it is possible to synthesize full-length molecules with the sequences:

SEC A: maESEKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK (SEQ ID NO: 10)

(in which ma designates meracaptoacetyl coupled with the N-terminal end of the polypeptide); Y

SEC B: AEAKYAKEMW IAWEEIRNLP NLNGWQMTAF IAKLLDDPSQ SSELLSEAKK LNDSQAPKC (SEQ ID NO: 11);

while conventional synthesis did not produce the peptide with SEC A.

Conventional synthesis of SEC C: AENKFNKEMW IAWEEIRNLP NLTGWQMTAF IASLLDDPSQ SANLLAEAKK LNDAQAPK (SEQ ID NO: 12), which is similar to SEC B but contains the amino acids of the original framework, resulted in a very impure preparation and low peptide production.

The introduction of new serine or threonine residues also allows the use of isoacyl dipeptides, which is an alternative to pseudoproline to increase synthetic efficiency by reducing aggregation during peptide synthesis (Sohma et al, Tetrahedron Lett. 47: 3013, 2006) . Several such basic Novabiochem components are available from Merck Biosciences AG.

Basis

Peptide synthesis of the HER2 binding molecule ZHER2: 342 (disclosed in WO 2005/003156 as ZHER2: 107, and sometimes also called Z00342), as well as DOTA coupling with the N-terminal end for this molecule is possible and is described in the literature (Orlova A et al (2006) Cancer Research 67: 21782186). However, a large variation in peptide production was observed after synthesis. The difficulties in reproducibly synthesizing the peptide can be related to both the length of the peptide and the primary amino acid sequence. In addition, long peptides with the reactive groups of the side chains of amino acids still protected can generate unfavorable secondary structures, for example beta sheets that may disturb solid phase peptide synthesis (Quibell M and Johnson T in Fmoc Solid Phase Peptide Synthesis-A Practical Approach, WC Chan, PD White Eds, Oxford University Press 2000: 115135). One way to avoid secondary structure formation during peptide synthesis is the use of pseudoprolines. Pseudoprolines, with the simplified abbreviation Fmoc-Xxx-Yyy-OH, can be used if the amino acid Yyy is serine, threonine or cysteine. These pseudoprolines have a proline type structure closed with the side chain linked to the main chain and can become the normal amino acid structure by acid treatment (Haack T and Mutter M (1992) Tetrahedron Lett 33: 1589-1592). Pseudoprolines are commercially available for 14 amino acids in the Xxx position (all naturally occurring amino acids except Arg, Cys, His, Met, Pro, Thr) together with serine or threonine in the Yyy position.

The precursor molecule ZHER2: 342 does not have threonine or cysteine in the primary sequence. The serine is only in positions 33, 39 and 41. A pseudoproline precursor is available only for serine 41 (Q40-S41). For the two other serines, the amino acid in the Xxx position avoids the use of pseudoproline, since there are no precursors available (R32-S33 and P38-S39).

Mutations introduced into the polypeptides comprised in the population according to the invention are directed to, but without restriction, facilitate peptide synthesis. Especially mutations at positions 23, 42, 46 and 54, that is [N23T], [A42S], [A46S] and [A54S] can have the ability to solve two of the problems identified in SPPS: they allow the use of pseudoprolines and the critical region around amino acid positions 21 to 26 is changed to position 23 replacing asparagine with threonine.

Synthesis Strategy 1

The SEC A amino acid sequence was assembled into a Fmoc-Lys (Boc) -Wang polystyrene resin in a fully automatic peptide synthesizer. This resin is highly suitable for peptide formation with the Fmoc strategy. 57 amino acids (with appropriate side chain protection) were coupled into the resin. In the last stage, the coupling of mercaptoacetic acid protected with S-trityl was performed manually.

Stage 1: Synthesis of Solid Phase Peptides

The Fmoc-Lys (Boc) -Wang polystyrene resin was transferred to an SPPS reactor with a stirrer. Synthesis was then started with Fmoc deprotection of the resin, followed by a coupling procedure with Fmoc-Pro-OH according to the general description provided below. This step was followed again by an Fmoc deprotection and subsequent coupling of the amino acid derivatives according to the sequence. After final washing of the resin with isopropylether (IPE), the peptide resin was dried in a desiccator under reduced pressure.

Both conventional Fmoc peptide synthesis and synthesis were performed using pseudoprolines in four positions. For conventional peptide synthesis, only Fmoc amino acids were used. For the synthesis of alternative peptides, apart from Fmoc amino acids, the following pseudoprolines were used: Fmoc-Leu-Thr-OH at position 22-23, FmocSer-Ser-OH at position 41-42, Fmoc-Leu-Ser-OH at position 45-46 and Fmoc-Asp-Ser-OH at position 53-54.

Fmoc Checkout Procedure

The resin was also treated with 20% piperidine in N-methyl-2-pyrrolidone (NMP) to achieve cleavage of the N-a-Fmoc protecting group. The resin washing was then carried out with NMP.

Coupling Procedure

Automatic coupling of amino acid derivatives Pro57 to Glu1. Up to 3 equivalents of the Fmoc-AA derivative were dissolved in NMP. For coupling, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in dimethylformamide (DMF) and sym-colidine (2,4,6-trimethylpyridine) were added in NMP The resulting solution was mixed at room temperature before pouring it into the resin. NMP was used as solvent. After a coupling time of at least 15 minutes at 60 ° C, the resin was washed with NMP.

After each coupling procedure, a repeat of the coupling takes place with 2- (1H-7azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TATU) in DMF as coupling reagent and with dichloroethane as solvent automatically, followed by acetic anhydride protection.

Stage 2: Mercaptoacetic Acid Coupling

Acylations were performed with 5 molar equivalents of amino acid, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorphosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) and 10 equivalents of N-ethyldiisopropylamine ( DIEA, from Lancaster Synthesis, Morecambe, England). S-trityl mercaptoacetic acid was from AnaSpec Inc (San Jose, CA, United States).

Stage 3: Excision of the resin including excision of the remaining protection groups

The peptide resin was treated with trifluoroacetic acid (TFA) in the presence of purified water, ethanedithiol (EDT) and triisopropylsilane (TIS). After about 2 hours of cleavage time at room temperature, the reaction mixture was cooled to about 0 ° C, and ammonium iodide and dimethyl sulfide were added to reduce oxidized methionine. After an additional cleavage time of 60 minutes at about 0 ° C, the iodine formed with ascorbic acid was reduced. After filtering off the product, it was precipitated in cold IPE, filtered off again, washed with IPE and dried under reduced pressure.

HPLC purity analysis

The purity of 58 amino acids in length and some intermediates peptides was determined by reverse phase HPLC using a Vydac 218 TP54 column (5 µm, 250 x 4.6 mm) and 0.1% TFA, 1% acetonitrile in H2O and 0.1% TFA in acetonitrile as solvent A and B respectively. The column oven temperature was set at 35 ° C. The column was eluted with a gradient of 15 to 45% of solvent B in 30 minutes or with a gradient of 20 to 50% of B in 30 minutes. UV detection was at 220 nm. Purity was calculated by area normalization.

Results

The performance and purity of the molecule were analyzed with the SEC A sequence, synthesized with or without the use of pseudoprolines, by analytical reverse phase chromatography. To follow the progress of the synthesis, a small part of the synthesis resin was taken after several stages of coupling and analyzed for the presence, purity and yield of the desired peptide intermediate. Figure 5 shows the HPLC analysis of the peptide intermediate 41 amino acids in length (amino acids 18-58). At this stage of the peptide synthesis, a clear and predominant peptide peak was identified with the correct sequence (TA = 15: 33 min, yield 49%) if the synthesis was performed using pseudoprolines (Figure 5A). Conventional Fmoc synthesis, however, resulted in a large number of small peptide peaks and two main peaks with similar size, but poor performance. One of these two peaks (TA = 20.82 min) was identified as the intermediate peptide with the correct sequence (aa 18-58) (Figure 5B). The full length peptide (amino acids 1-58) was obtained only if the synthesis was performed using pseudoprolines. Figure 6A shows a single product peak with a final peptide yield of 26%. Conventional Fmoc synthesis, however, did not produce the final peptide product. Analysis of the 49 amino acid length intermediate (amino acids 10-58) of the conventional synthesis revealed that the desired intermediate could not be detected and the synthesis was suspended (Figure 6B).

Synthesis Strategy 2

Two molecules were assembled using Fmoc's strategy in a fully automatic peptide synthesizer with an integrated microwave oven.

The 59 amino acid residues of SEC B, based on the framework sequence of the invention, were assembled (with appropriate side chain protection) in an Fmoc-Cys (Trt)-Wang LL polystyrene resin.

The 58 amino acid residues of SEC C, based on the original Affibody® molecule framework, were assembled (with appropriate side chain protection) in a Fmoc-Lys (Boc)-Wang LL polystyrene resin.

Wang LL resin is highly suitable for peptide formation with the Fmoc strategy.

Stage 1: Synthesis of solid phase peptides

The polystyrene resin was automatically transferred to an SPPS reaction vessel by the synthesizer (Liberty, CEM Corporation, NC United States). Synthesis was then initiated with Fmoc deprotection of the resin, followed by a coupling procedure with the following Fmoc protected amino acid (Fmoc-AA) in accordance with the general description provided below. This step was followed again by an Fmoc deprotection and subsequent coupling of the amino acid derivatives according to the sequence. After the final washing of the resin with dichloromethane (DCM), the peptide resin was dried under reduced pressure. The complete SEC C peptide was prepared by conventional Fmoc peptide synthesis, while pseudoprolines were used in the positions in SEC B in which this was made possible by the improvements made to the framework. The following pseudoprolines were used: Fmoc-Ser-Ser-OH at position 41-42, Fmoc-Leu-Ser-OH at position 45-46 and Fmoc-Asp-Ser-OH at position 53-54.

Fmoc Checkout Procedure

The resin was treated with 5% piperazine in NMP, with microwave irradiation, to achieve cleavage of the N-a-Fmoc protecting group. The resin washing was then carried out with NMP.

Coupling Procedure

Automatic coupling of amino acid derivatives Cys59 to Ala1 (for SEC B) and Lys58 to Ala1 (for SEC C). Up to 5 equivalents of Fmoc-AA were dissolved in NMP. For coupling, O- (benzotriazolN, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), N, N'-diisopropylethylamine (DIPEA) in NMP were added to the resin at a molar ratio of 1: 1: 1: 2 (AA / HBTU / HOBt / DIPEA) The mixture was stirred by bubbling nitrogen gas through the bottom of the reaction vessel, after a coupling time of at least 5 minutes at 75-80 ° C with added energy using microwave irradiation, the resin was washed with NMP.

After each coupling procedure, a protection with automatic acetic anhydride was performed.

Stage 2: Excision of the resin that includes excision of the remaining protection groups.

The peptide resin was treated with trifluoroacetic acid (TFA) in the presence of purified water, ethanedithiol (EDT) and triisopropylsilane (TIS). After about 2 hours of cleavage at room temperature, the cleavage mixture was filtered and the resin was rinsed with water / 95% pure TFA. The filtrate was added slowly to cooled methyl tert-butyl ether (MTBE). The precipitate was centrifuged and the MTBE was removed. The solid was resuspended in ether and the operation was repeated a total of three times. After the last ether removal, the solid was resuspended in water / 0.1% TFA, the remaining ether was allowed to evaporate and the solution was frozen before lyophilization.

Mass and purity analysis by HPLC-MS

The purity of the peptides was determined by high performance liquid chromatography and in-line mass spectrometry (HPLC-MS) using an Agilent 1100 HPLC / MSD equipped with electrospray ionization (IEN) and a single quadrupole. HPLC was processed using a Zorbax 300SB C18 column (3.5 µm, 150 x 4.6 mm) and water / 0.1% TFA and 0.1% TFA / acetonitrile (ACN) as solvent A and B respectively. The temperature of the column oven was set at 30 ° C. The column was eluted with a solvent gradient B of 15 to 55% in 40 minutes. UV detection was at 220 nm. Purity was calculated by area normalization. The software used for mass analysis and evaluation was ChemStation Rev. B. 02.01. (Agilent).

Results

The yield and purity of the SEC B and SEC C molecules was analyzed by analytical reverse phase chromatography. Full-length peptides were obtained in both syntheses, however with much higher yield for SEC B. Figure 7A shows, for SEC B, a main product peak (TR = 41.48 min) with the expected mass and a 15% final peptide yield. It was found that an additional peak (TR = 41.21 min) with a yield of 8% had a mass that was 72 Da higher than the expected mass of the full length product. It is believed that this is due to a secondary reaction in the Cys59 amino acid residue. Depending on the type of secondary reaction, this may occur during synthesis or during cleavage of the resin peptide. By optimizing the cleavage and / or synthesis protocol, this secondary reaction could be minimized and thus the yield increased, in this case to a total yield of 23%.

Conventional Fmoc synthesis of SEC C, however, resulted in a large number of small peptide peaks (Figure 7 B). One of the main peaks (RT = 43.60 min) was identified as the full length peptide with the expected mass. The yield of this product was 4%.

Example 6 - Immunogenicity of original polypeptide variants and of the invention

Summary

In this example, the immunogenicity of an original polypeptide variant and one of the invention was compared in vivo. Dimeric molecules were administered to rats, and specific antibody responses were determined in an anti-drug antibody (ADA) assay. The molecule with the framework mutations introduced according to the invention has a lower and delayed antibody response compared to the original Z variant.

Cloning and production of polypeptides

Two Taq-polymerase specific binding polypeptides fused with the albumin binding domain ABD035 (Jonsson et al (2008) Protein Eng Des Sel 8: 515-27) were used in the study:

one.

 (Z01154) 2-ABD035: original frame

2.

 (Z03229) 2-ABD035: framework of the invention.

PCR amplified and hybridized fragments of Z01154 and Z03229 were cloned with Accl protrusions as dimers in the expression vectors derived from AccET digested pET (Novagen) pAY492 and pAY1450, respectively. The resulting vectors were digested with Accl-Notl and ligated with ABD035 fragments that had been amplified by PCR with Accl and Notl projections, generating the pAY1827 constructs (encoding MGSSLQ- [Z01154] - [Z01154] -VD- [ABD035]) and pAY2292 (encoding MGSSLQ- [Z03229] - [Z03229] -VD- [ABD035]). Plasmids were transformed into competent E. coli BL21 (DE3) cells and proteins were produced by fermentation, essentially as described in Example 3. The pelleted cells were suspended in [25 mM Tris-HCl, 200 mM NaCl, EDTA 1 mM, Benzonase® 25 U / ml (Merck, No. 1.01654.0001), pH 8.0] and were broken by sonication on ice. The clarified supernatants were loaded on a column packed with CNBr activated Sepharose (GE Healthcare, No. 17-0981-03) internally coupled with human serum albumin. The column was pre-equilibrated in 1 x TST [25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.05% Tween 20, pH 8.0]. After application of the sample, washing was performed with 1x TST followed by 5 mM NH4Ac pH 5.5, until no reduction in the Abs280 signal was observed. Bound proteins were eluted with 0.5 M Hac, pH 2.8. Eluted samples were supplemented with acetonitrile at a final concentration of 2% and were further purified by reverse phase chromatography on a Resource RPC column (GE Healthcare, No. 17-1182-01). [2% acetonitrile, 0.1% TFA in water] was used as the execution buffer and the samples were eluted using a linear gradient of 0-50% of [80% acetonitrile, 0.1% TFA in water] over 25 volumes of column. Buffer exchange was performed to [5 mM sodium phosphate, 150mM NaCl, pH 7.2] using a HiPrep 26/10 Desalination column (GH Healthcare, No. 17-5087-01). Sample purity was verified by SDS-PAGE and LC / MS analysis as described in Example 3. Traces of endotoxins were removed on an AffinityPak Detoxi-Gel endotoxin withdrawal gel (Thermo, No. 20344) of According to the manufacturer's instructions. No endotoxins were detected in the LAL gel coagulation assays by APL (Apoteket Produktion & Laboratorier AB, Sweden). The samples were without soluble aggregates as verified by size exclusion chromatography performed on a Superdex 75 10/300 column (GE Healthcare, No. 17-5174-01) using 1 x PBS as the execution buffer, a flow rate of 0 , 5 ml / min and a sample volume of 100 µl with a concentration of 1 mg / ml.

Administration and sampling schemes

The animal study was conducted in Agrisera AB (Vännäs, Sweden) with permission from the local animal ethics committee. Sprague Dawley female rats were injected into three groups subcutaneously (Z01154) 2-ABD035, (Z03229) 2-ABD035 or a buffer control as outlined in Table 9. Injections were provided on days 0, 4, 7 , 14, 21 and 28. Blood samples of 250 µl were collected from each animal on day -1 (before serum) and on days 6, 13, 20 and

35. All animals were sacrificed on day 35. The blood samples collected were allowed to clot overnight at 4 ° C and the sera obtained were stored at -20 ° C until analysis.

Table 9. Sample administration scheme

Group Average molecule number of mg / animal / ml injection / animal / animal injection administration

1 8 (Z01154) 2-ABD035 s.c. 0.125 0.1

2 8 (Z03229) 2-ABD035 s.c. 0.125 0.1

3 4 Buffer control: s.c. -0.1 5 mM sodium phosphate, 150 mM NaCl, pH 7.2

Anti-drug antibody (ADA) assay

10 To analyze the presence of anti-(Z01154) 2-ABD035 and anti-(Z03229) 2-ABD035 antibodies, three types of ELISA analysis were performed. All samples were initially screened for the presence of reactive antibodies followed by a confirmation test to verify specificity. Serum samples with specific antibodies against Z variants were subsequently titrated to quantify the titer of anti-(Z01154) 2-ABD035 and anti-(Z03229) 2ABD035 antibodies.

15 For the examination of serum samples, ELISA plates (96-well half-area plates, Costar, No. 3690) were coated overnight with (Z01154) 2-ABD035 or (Z03229) 2-ABD035 diluted in coating buffer (Sigma, No. C3041) to a final concentration of 2 µg / ml. 50 µl of the coating solution was added per well and the plates were incubated overnight at 4 ° C. The plates were washed twice manually with deionized water and subsequently blocked for 2 hours with PBS-Casein 100

20 µl / well (PBS with 0.5% Casein (Sigma, No. 8654)). The blocking solution was removed and serum samples (50 µl / well) diluted 1:50 in blocking buffer were added. After 1.5 hours of incubation at RT, the plates were washed in an automatic ELISA scrubber (Scanwasher 300, Scatron) with PBST (PBS with 0.05% Tween 20 (Acros Organics, No. 233362500)). To detect rat antibodies against Z variants, 50 µl was added per well of HRP-conjugated anti-rat IgG (Southern Biotech, No. 3050-05), diluted 1: 6000 in PBS-Casein.

After 1 hour of incubation, the plates were washed as described above and 50 µl / well of substrate solution (Immunopure® TMB, Pierce, No. 34021) was added. The plates were incubated at RT in the dark, and color development was stopped after 15 minutes with 50 µl / well of 2M H2SO4 (VWR, No. 14374-1). The plates were read at 450 nm in an ELISA reader (Victor3, Perkin Elmer).

The ELISA procedure described above was also used for confirmation and ELISA assays.

30 assessment, but with some alterations. For the confirmation test, serum samples were diluted 1:50 in PBS-Casein or in PBS-Casein that included 1 µg / ml of respective polypeptide variant. Serum samples with a DO signal reduction of 45% or more were considered to contain specific antibodies against Z variants. For titration assay, serum samples 1:50 were diluted in PBS-Casein and then in series of dilutions 2 times or 5 times until they crossed the specific plate cutoff to allow

35 a titration value for the sample is calculated.

During the trial, the following key parameters were determined:

Minimum dilution: 1:50 Non-specific background (NSB): DO450 of a group of normal rat serum (Sprague Dawley rats, Scanbur) used as dilution matrix and included in each plate throughout the analysis

40 Test cut-off point: DO450 average of normal rat sera of 30 individuals plus 1,645 times the standard deviations from the mean. A value of 0.11 was obtained for both (Z01154) 2-ABD035 and (Z03229) 2-ABD035. Normalization factor Cut-off point of the test divided by the average OD450 of the NSB: 1.87 and 1.86 for (Z01154) 2-ABD035 and (Z03229) 2-ABD035, respectively.

During the sample analysis, the plate-specific cut-off point was then determined as: Mean of OD450 of plate-specific NSB, multiplied by the normalization factor.

Serum from rats (hyperimmunized Sprague Dawley rats, Agrisera) was used which had been confirmed to contain antibodies against the two polypeptide variants to prepare positive control samples (CP) included in each plate throughout the analysis: High CP: positive control serum diluted 1: 4 in the matrix before the minimum dilution in PBS-Casein. This CP has high OD values above the test cut-off point / plate. Low CP: positive control serum diluted 1: 300 in matrix before the minimum dilution in PBS-Casein. This CP has OD values that are just above the test cut-off point / plate.

The Low CP and High CP values were used to prepare the LoQC1-5 and HiQC1-5 titration quality controls. The Low CP and High CP were diluted 1:50 in PBS-Casein to obtain LoQC1 and HiQC1 respectively. These were further diluted in PBS-Casein to obtain LoQC2 (1: 100), LoQC3 (1: 200), LoQC4 (1: 400) and LoQC5 (1: 800), and HiQC2 (1: 250), HiQC3 (1 : 1250), HiQC4 (1: 6250) and HiQC5 (1: 31250), respectively.

Titration values were calculated using GraphPad Prism 5 (GraphPad Software Inc). Briefly, OD450 values were plotted against logarithmic dilution and sample titration was defined as logarithmic dilution at the specific plate cutoff.

Results

The in vivo comparison between the original ((Z01154) 2-ABD035) and the invention ((Z03229) 2-ABD035) molecules showed that the molecule of the invention was less immunogenic. The response varied considerably among individuals and increased over time. Titration could be determined in three individuals who received the original molecule compared to two individuals who received the molecule of the invention. The actual titration was also lower in the group that received the molecule of the invention (Table 10). The reason to see that few animals develop an antibody response may be due to the fused ABD molecule, which has previously been shown to reduce the immunogenicity of a fused polypeptide (see for example, WO 2005/097202).

Table 10. Immune responses in rats to which an original polypeptide variant or one of the invention was provided

Group 1 (Z01154) 2-ABD035 n = 8

Group 2 (Z03229) 2-ABD035 n = 8 Group 3 buffer control n = 4

Time (days)

Specific answer (number of animals) Average Degree ± DT Specific answer (number of animals) Average Degree ± DT Specific answer (number of animals) Average Degree ± DT

-one

0 - 0 - 0 -

6

0 - 0 - 0 -

13

one 2.9 0 - 0 -

twenty

2 3.4 (± 1.0) one 2.1 0 -

27

3 2.7 (± 1.0) one 2.4 0 -

35

2 3.4 (± 1.3) 2 2.8 (± 0.1) 0 -

SEQUENCE LIST

<110> AFFIBODY AB

<120> POLYPEPTIDE VARIANTS

<130> 21039128

<150> EP07150394

<151>

<150> US 61 / 009,171

<151>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 48

<212> PRT

<213> Artificial

<220>

<223> artificial population of polypeptide variants

<220>

<221> misc_feature

<222> (2) .. (4)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (6) .. (7)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (10) .. (11)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (17) .. (18)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (20) .. (21)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (25) .. (25)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (28) .. (28)

<223> xaa can be any naturally occurring amino acid

<400> 1 <210> 2

<211> 53

<212> PRT

<213> Artificial

<220>

<223> artificial population of polypeptide variants

<220>

<221> misc_feature

<222> (7) .. (9)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (11) .. (12)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (15) .. (16)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (22) .. (23)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (25) .. (26)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (30) .. (30)

<223> xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (33) .. (33)

<223> xaa can be any naturally occurring amino acid

<400> 2

Claims (21)

one.

 A population of polypeptide variants based on a common framework, each polypeptide comprising in the population the amino acid sequence of framework EXXXAXXEIXXLPNLTXXQX XAFIXKLXDD PSQSSELLSE AKKLNDSQ, (SEQ ID NO: 1) in which each X individually corresponds to an amino acid residue that varies in the population.

2.

 A population of polypeptide variants according to claim 1, wherein said sequence of framework amino acids is

in which each X corresponds individually to an amino acid residue that varies in the population. A population according to claim 1 or 2, comprising at least 1 x 104 unique polypeptide molecules.

Four.

 A population of polynucleotides, characterized in that each member thereof encodes a member of a population of polypeptides according to any one of claims 1-3.

5.

 A combination of a population of polypeptides according to any one of claims 1-3 with

A population of polynucleotides according to claim 4, wherein each member of said polypeptide population is physically or spatially associated with the polynucleotide encoding that member by means of genotype-phenotype coupling. 6. A method for selecting a desired polypeptide having an affinity for a predetermined target of a population of polypeptides, comprising the steps of: 20 (a) providing a population of polypeptides according to any one of claims 1-3; (b) bringing the population of polypeptides into contact with the predetermined target under conditions that allow specific interaction between the target and at least one desired polypeptide having an affinity for the target; Y (c) selecting, based on said specific interaction, the at least one desired polypeptide from the remaining population of polypeptides. 7. A method according to claim 6, wherein step (a) comprises the preparatory steps of providing a population of polynucleotides according to claim 4 and expressing said population of polynucleotides to produce said population of polypeptides, in the that each member of said polypeptide population is physically or spatially associated with the polynucleotide encoding that member by means for 30 genotype-phenotype coupling. 8. A method for isolating a polynucleotide encoding a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

-

selecting said desired polypeptide and the polynucleotide encoding it from a population of

polypeptides using the method according to claim 7; and isolating the separated polynucleotide in this way that encodes the desired polypeptide. 9. A method for identifying a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

-

isolating a polynucleotide encoding said desired polypeptide using the method according to claim 8; Y

Sequencing the polynucleotide to establish by deduction the amino acid sequence of said desired polypeptide. 10. A method for selecting and identifying a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps of: (a) synthesizing each member of a population of polypeptides according to any one of claims 1-3 in a separate vehicle or bead;

(b)

 select or enrich the vehicles or beads based on the interaction of the polypeptide with the predetermined target; Y

(C)

 Identify the polypeptide by protein characterization methodology.

11. A process for the production of a desired polypeptide having an affinity for a predetermined target, comprising the steps of:

-

isolate and identify a desired polypeptide using the method according to claim 9; or

selecting and identifying a desired polypeptide using the method according to claim 5; Y

-

producing said desired polypeptide.

12. A process for the production of a desired polypeptide having an affinity for a predetermined target, comprising the steps of: (a1) isolating a polynucleotide encoding said desired polypeptide using the method according to claim 8; or (a2) roll back a polypeptide identified using the method according to claim 10; Y (b) after (a1) or (a2), express the polynucleotide isolated in this way to produce said desired polypeptide, 13. A process for the production of a first polypeptide based on a framework, comprising the steps 15 to provide a second polypeptide having affinity for a predetermined target being said second polypeptide based on an original SpA-derived framework, and mutating the amino acids of the original framework to generate the first polypeptide comprising the amino acid sequence of the framework 20 EXXXAXXEIXXLPNLTXXQX XAFIXKLXDD PSQSSELLSE AKKLNDSQ (SEQ ID NO: 1), in which each X individually corresponds to an amino acid residue that is conserved from the second polypeptide. 14. A method according to claim 13, wherein said amino acid sequence of the framework is In which each X corresponds individually to an amino acid residue that is conserved from the second polypeptide. 15. A method according to any one of claims 13 and 14, wherein said original framework comprises an amino acid sequence of the framework selected from EXXXAXXEIXXLPNLNXXQXXAFIXSLXDD PSQSANLLAE AKKLNDAQ (SEQ ID NO: 3) and 30 in which each X corresponds individually to any amino acid residue.

16.

A method according to any one of claims 13-15, wherein said original framework derives from SpA domain B and comprises a G29A mutation, corresponding to A at position 22 in SEQ ID NO: 1 and A at position 27 in SEQ ID NO: 2.

17.

A polypeptide having affinity for a predetermined target, which can be obtained by a method of

According to any one of claims 13-16 and comprises the first amino acid sequence of the framework EXXXAXXEIXXLPNLTXXQX XAFIXKLXDD PSQSSELLSE AKKLNDSQ, (SEQ ID NO: 1), wherein each X corresponds to a randomly selectable amino acid residue in a second polypeptide based on an amino acid sequence of the original framework and in which said second polypeptide has affinity 40 for said predetermined target. 18. A polypeptide according to claim 17, wherein said first amino acid sequence of the framework is wherein each X corresponds to a randomly selectable amino acid residue in a second polypeptide based on an amino acid sequence of the original framework and wherein said second polypeptide has affinity for said predetermined target.

19.

Use of a polypeptide according to any one of claims 17-18 as a detection reagent, capture reagent or separation reagent.

twenty.

 A polypeptide according to any one of claims 17-18 for use in therapy.

twenty-one.

 A polypeptide according to any one of claims 17-18 for use as a diagnostic agent.

22

 A polypeptide according to any one of claims 17-18 wherein said target is selected from TNF-a, insulin and Taq polymerase.